Alteration of gut microbiota in high‐fat diet‐induced obese mice using carnosic acid from rosemary

Abstract Rosmarinus officinalis (rosemary) is widely used as a food ingredient. Rosemary extract (containing 40% carnosic acid) exhibited potent antiobesity activity. However, the relationship between carnosic acid (CA) and changes in the gut microbiota of high‐fat diet (HFD)‐induced obese mice has not been fully investigated. C57BL/6 mice were fed a normal diet, an HFD, or an HFD containing 0.1% or 0.2% CA for 10 weeks. CA exhibited promising antiobesity effects and caused marked alterations in the gut microbiota of HFD‐induced obese mice. CA caused the prevalence of probiotics and functional bacteria, including Akkermansia muciniphila, Muribaculaceae unclassified, and Clostridium innocuum group, and inhibited diabetes‐sensitive bacteria, including Proteobacteria and Firmicutes. The ratio of Firmicutes to Bacteroidetes was regulated by CA in a dose‐dependent manner, decreasing it from 13.22% to 2.42%. Additionally, CA reduced bile acid‐metabolizing bacteria, such as Bilophila, Clostridium, Lactobacillus, and Leuconostoc. The results of the linear discriminant analysis and effect size analysis indicated that CA attenuated the microbial changes caused by HFD. The high CA (HCA) group (HFD containing 0.2% CA) exhibited a greater abundance of Verrucomicrobiae (including Akkermansia muciniphila, genus Akkermansia, family Akkermansiaceae, and order Verrucomicrobiales), Eubacterium, and Erysipelatoclostridium, and the low CA (LCA) group (HFD containing 0.1% CA) exhibited a greater abundance of Eisenbergiella, Intestinimonas, and Ruminococcaceae. Our results demonstrate that the antiobesity effects of CA might be strongly related to its prebiotic effects.

lipid accumulation in hepatocytes by activating the AMPK/PPAR and EGFR/MAPK pathways (Wang et al., 2012;Zhao et al., 2015). The gut microbiota plays an important role in the bioactivity of many natural products (Cotillard et al., 2013). For example, the antidepressant effects of rosemary extract are mediated by rebalancing the gut microbiota (Guo et al., ). Carnosic acid (CA) is the major active component of rosemary and has been extensively studied . It has been reported that rosemary extracts (containing 40% CA) change the microbiota composition of female Zucker rats, but only a few members of the phyla Firmicutes, Bacteroidetes, and Actinobacteria were studied (Romo-Vaquero et al., 2014). Thus, the modulatory effect of CA on gut microbiota needs to be investigated further.
C57BL/6 mice were fed an HFD supplemented with or without CA for 10 weeks. To our knowledge, this is the first report on the modulating effect of CA on gut microbiota in HFD-induced obese mice.

| Plant material
The leaves of R. officinalis were collected from Yulin (Guangxi, China) in

| High-performance liquid chromatography (HPLC)
A Waters H-Class instrument (Waters) was used for HPLC analysis.
Chromatographic separation was performed on a Hypersil GOLD column (1.9 μm, 100 mm × 2.1 mm) using 0.1% formic acid solution and acetonitrile as the mobile phase.

| Extraction and isolation
Dried leaves of R. officinalis (12.0 kg) were percolated with ethanol (3 × 25 L) to obtain a crude extract. The extract (561.5 g) was partitioned into water (2.5 L) and extracted successively with hexane, ethyl acetate, and n-butanol. The hexane fraction was subjected to repeated CC to obtain carnosic, carnosol, and rosmarinic acids. Their structures were identified using mass spectrometry and nuclear magnetic resonance spectroscopy. The purities of CA, carnosol, and rosmarinic acid were determined using an ACQUITY UPLC H-Class system (Waters Co.) with a C-18 column (Thermo, 1.9 μm, 4.6 mm × 150 mm), and were found to be ≥95%.
The protocol and experimental procedures for this study were approved by the Ethics Committee for Animal Experimentation of Wuyi University, and they followed the National Institute of Health's Guide for the Care and Use of Laboratory Animals.
The mice were divided into the following groups (n = 6 for each group): HFD group (fed with HFD containing 60% calories from fat), low CA (LCA) group (fed with HFD containing 0.1% CA), high CA (HCA) group (fed with HFD containing 0.2% CA), carnosol (CO) group (fed with HFD containing 0.2% carnosol), rosmarinic acid (RA) group (fed with HFD containing 0.2% rosmarinic acid), and the normal diet (ND) group (fed with normal diet containing 10% fat calories). A high-fat diet (product number: TP23300) and a normal diet (product number: TP233020) were purchased from Trophic Animal Feed High-Tech Co. Ltd. Food consumption was monitored daily and body weight was measured weekly. The mice were housed under a light/dark cycle (12/12 h) at an ambient temperature of 22 ± 2°C with constant humidity and given water and food ad libitum. Feces were collected three times a week and stored at a temperature of 80°C in a freezer. After 10 weeks, all the mice were sacrificed and the adipose tissue (epididymal, retroperitoneal, and mesenteric), liver, kidney, and spleen were removed, weighed, placed in vials immediately, and frozen in liquid nitrogen.

| DNA extraction and 16S rRNA gene sequence analysis
The methods for DNA extraction, sequencing, and data analysis were as described previously by Segata et al. (Segata et al., 2011). Briefly, total DNA was extracted using an E.Z.N.A. ® Stool DNA kit (D4015, Omega, Inc.), according to the manufacturer's instructions. Amplicon sequencing was performed using the Illumina MiSeq platform. After merging paired-end reads (FLASH) and quality control (fqtrim, V0.94), sequences with ≥97% similarity were regarded as the same operational taxonomic units (OTUs) using Vsearch (v2.3.4). The OTUs were classified using the Ribosomal Database Project software, and OTU abundance data were normalized with a standard sequence number.
The analyses were performed by LC-Bio Tech Co., Ltd.

| Statistical analysis
Experimental data are expressed as the mean ± standard deviation.
The statistical significance was calculated using a one-way analysis of variance (ANOVA) followed by a post hoc test. Linear discriminant analysis effect size (LEfSe) analysis was performed using the LEfSe software (http://hutte nhower.sph.harva rd.edu/galax y/) with a logarithmic discriminant analysis (LDA) threshold score of 4.0.

| Extraction and isolation of compounds from rosemary
Rosemary is rich in bioactive phenolics, and HPLC analysis has shown that the major bioactive phenols in rosemary comprise of CA (6.30 mg/g), carnosol (3.61 mg/g), and rosmarinic acid (1.92 mg/g) of the dry weight of rosemary ( Figure 1, Table 1). The ethanol extract of rosemary was subjected to repeated CC to yield 12 g CA, 5 g carnosol, and 4 g rosmarinic acid.

| Carnosic acid reduced body weight gain and food efficiency
Previous studies found that rosemary extract exhibits promising antiobesity effects (Zhao et al., 2015). To explore the mechanism of the antiobesity activity of rosemary, CA, carnosol, and rosmarinic acid were screened for antiobesity effects in HFD-induced obese mice. The results demonstrated that carnosol and rosmarinic acid did not significantly affect body weight in mice, whereas CA significantly affected body weight gain after 10 weeks of feeding (p < .05, Table 2). In addition, CA did not decrease the weight of the spleen and liver but alleviated the weight gain of the kidney caused by the HFD. Moreover, HCA caused a significant reduction in food efficiency (p < .05) compared with that caused by the LCA and HFD groups.

| Carnosic acid modulated structural composition of the gut microbiota
CA exhibited significant antiobesity effects, but the relationship between the antiobesity activity of CA and its modulating effect on the gut microbiota has not yet been well investigated. Hence, 16S rRNA gene sequence analysis was performed.
The results of the alpha diversity analysis indicated that HFD feeding markedly reduced bacterial richness and diversity, as verified by the increased Simpson index and reduced Chao 1 and Shannon indices compared with those obtained for the ND group (Table 3). Nevertheless, HCA significantly ameliorated the diversity of gut microbes (p < .05).
At the phylum level, the microbial community was mainly composed of Firmicutes, Verrucomicrobia, Bacteroidetes, Proteobacteria, and Actinobacteria ( Figure 2a). The HCA and LCA groups had a higher abundance of Verrucomicrobia, Bacteroidetes, and Epsilonbacteraeota, and a lower abundance of Firmicutes, Proteobacteria, and Actinobacteria compared with that of the HFD group (p < .05, Table   S1). The ratios of Firmicutes/Bacteroidetes (F/B) for HFD, LCA, HCA, and ND were 13.22 ± 0.31, 7.08 ± 0.58, 2.42 ± 0.14, and 2.35 ± 0.50, respectively. The results indicated that the decrease in the F/B ratio caused by CA was dose dependent and that the HCA group could lower the F/B ratio to a level similar to that of the ND group.
In addition, the HCA and LCA groups had a significantly higher abundance of Gammaproteobacteria and Campylobacteria and a significantly lower abundance of Coriobacteriia, Bacilli, Saccharimonadia, Deltaproteobacteria, and Actinobacteria than that of the HFD group (p < .05, Table S1). Compared with the HFD group (5.63% ± 0.38%), Bacteroidia increased in a dose-dependent manner in the LCA and HCA groups (8.44% ± 0.76% and 20.26% ± 0.56%, respectively). In contrast, Clostridia decreased in a dose-dependent manner in the LCA and HCA groups (31.66% ± 5.49% and 12.64% ± 6.08%, respectively) compared with that of the HFD group (41.72% ± 5.99%).
The abundances of the top 30 genera in each group are shown in Figure 2c. The bacterial genera exhibiting the most significant increase in relative abundance after CA treatment were Akkermansia and Muribaculaceae unclassified. CA increased the relative abundance of these genera to a similar or higher level than that in the ND group ( Figure 3a). In contrast, CA decreased the relative abundance F I G U R E 1 HPLC traces of rosemary extracts of Bilophila, Clostridiales unclassified, and Clostridium in a dosedependent manner (Figure 3b and  Table S1).
In addition, this study discovered that CA significantly increased the abundances of Blautia and Bacteroides and significantly decreased Lactobacillus, Clostridium, and Leuconostoc (p < .05, Figure 3c,d, and Table S1). This result is consistent with that reported in previous literature (Romo-Vaquero et al., 2014).
Akkermansia muciniphila has been reported to exert an antiobesogenic effect and has been shown to significantly decrease obesity in animals. Owing to the notable bloom of Akkermansia, we compared the relative abundance of Akkermansia muciniphila among different groups. The results implied that HFD feeding significantly decreased the abundance of Akkermansia muciniphila compared with the effects of the normal diet, whereas Akkermansia muciniphila was significantly increased in the LCA and HCA groups (p < .05, Figure 3a and Table S1).

| Identification of key bacterial genera associated with weight regulation
Spearman correlation analysis was performed to identify key genera that were potentially relevant to the bodyweight of mice in the HFD and HCA groups. Four genera were significantly associated with a decrease in weight, namely: Akkermansia, Muribaculaceae unclassified, Escherichia-Shigella, and the Clostridium innocuum group. (Figure 2c, p < .05, Table S2). The phylotypes that were significantly associated with an increase in weight were the gen- *Data were expressed as the mean ± standard deviation (n = 6) and were analyzed using ANOVA followed by post hoc test. p < .05, compared with HFD group.

| Differential taxa in different fecal microbial communities
Linear discriminant analysis effect size (LEfSe) was performed to identify the enriched bacteria in each group. A total of 66 prokaryotic clades were screened out with an LDA threshold score of 4.0 ( Figure 4a).
Taxa with significantly higher abundance in the ND group ( Figure 4b)

| DISCUSS ION
The dosages of CA were selected based on previous studies that reported that 0.28% rosemary extract (containing 80% CA) could ameliorate obesity induced by an HFD in mice (Zhao et al., 2015), and that 0.5% rosemary extract (containing 40% CA) significantly changed the microbiota composition of female Zucker rats (Romo-Vaquero Verrucomicrobia Turnbaugh et al., 2006). The gut microbiota of obese humans and animals exhibit a higher F/B ratio than that of normal weight individuals. Our results indicated that CA increased the abundance of Verrucomicrobia and Bacteroidetes.
Moreover, CA reduced the abundance of Proteobacteria and the F/B ratio in a dose-dependent manner. The reduced F/B ratio suggests that the changed microbial components might lead to a lower efficacy for energy harvesting. The reduced Proteobacteria levels suggested that CA could alleviate the microbial disorder caused by HFD, since the prevalence of Proteobacteria was recognized as a signature for microbial dysbiosis in the obese microbiome (Shin et al., 2015).
The interaction between bile acid and the gut microbiome is well-known (al-Sereiti et al., 1999). Previous reports state that the increase in Bilophila is associated with an HFD and bile acid metabolism (David et al., 2014). In addition, decreased intestinal bile acid-related microbes, including Lactobacillus, Lactococcus, and Clostridium, increase the levels of ileal conjugated bile acids, which in turn inhibit the intestinal FXR-FGF15 signaling pathway, resulting in reduced hepatic cholesterol and lipogenesis (Huang et al., 2019). In this study, The abundance of Akkermansia muciniphila in healthy individuals is higher than that in obese individuals, and it has been identified as

F I G U R E 3
The relative abundance of Muribaculaceae unclassified, Akkermansia, Akkermansia muciniphila (a), Bilophila and Clostridiales unclassified, Clostridium (b), Eubacterium, Clostridium innocuum group, Bacteroides (c), Lactobacillus, and Leuconostoc (d), under different treatment. *p < .05, compared to HFD group beneficial bacteria (Everard et al., 2013). Administration of Akkermansia muciniphila could reverse metabolic disorders caused by HFD, such as insulin resistance, adipose tissue inflammation, and fat mass gain. The increase in Akkermansia muciniphila was the most significant in this study, suggesting that the prebiotic effect of Akkermansia muciniphila is crucial for the antiobesity activity of CA.

| CON CLUS ION
In conclusion, CA exhibits body weight-reducing effects and causes marked changes in the gut microbiota of HFD-induced obese mice.
The LEfSe indicated that CA attenuated the microbial changes caused by HFD. The modulating effect of CA on the gut microbiota was characterized by the promotion of probiotics and functional bacteria, including Akkermansia muciniphila and Bacteroidetes, and the inhibition of bile acid-metabolizing bacteria, including Bilophila, Clostridium, Lactobacillus, and Leuconostoc. Thus, the promotion of probiotics and functional bacteria and the inhibition of bile acidmetabolizing bacteria could be potential mechanisms by which rosemary elicits its antiobesity effects. Our results complement the current knowledge regarding the bioactivity of the phenolic constituents of rosemary.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

This study was approved by the Ethics Committee for Animal
Experimentation of the Wuyi University (Jiangmen, China).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author uponreasonable request.